Method and useful apparatus for preparing pharmaceutical compositions

ABSTRACT

The invention provides an apparatus and method for the preparation of a pharmaceutical composition comprising a complex of an antigen component and the corresponding antibody component in a pharmacologically acceptable carrier. The apparatus comprises a housing chamber for containing paramagnetic particles and a platform against which the housing chamber is releasably retained. The platform contains one or more magnets located adjacent to the housing chamber when the housing chamber is positioned on the platform. The platform is secured to a support which allows the platform, and thus the housing chamber, to be rotated between a vertical and a horizontal position. The method involves supplying a quantity of paramagnetic particles bearing the antigen component to the housing chamber, introducing into the housing chamber a physiological fluid which contains the antibody component, mixing the paramagnetic particles and the physiological fluid to form an antigen component and antibody component complex on the paramagnetic beads, removing the physiological fluid from the housing chamber, and eluting the antigen component and antibody component complex from the paramagnetic beads in a pharmaceutically acceptable eluting solution.

This is a continuation of U.S. Ser. No. 07/407,487 filed Sep. 14, 1989,now abandoned.

BACKGROUND OF THE INVENTION

The present invention is related to devices and methods of using thesame, to prepare pharmaceutical compositions comprising a complex of anantigen component and the corresponding antibody or inhibitor componentin a pharmacologically acceptable carrier. Specifically, the inventionconcerns a device and method of using the same, of preparing apharmaceutical composition by selectively isolating an antibody typecomponent from a physiological fluid, such as blood, plasma or otherbodily fluid in a pharmacologically acceptable carrier to which theantigen type compound may be added.

Pharmaceutical compositions containing antibody/antigen complexes havebeen suggested for numerous medical applications. For example, U.S. Pat.No. 4,740,371, issued to St. Remy et al on Apr. 26, 1988, disclosed acomplex useful for treating allergies. This complex includes thespecific allergen that causes the allergic reaction, and thecorresponding antibody for that allergen. While the antibodies may bederived from either the patient's or a donor's physiological fluids, thepreferred antibody is usually derived from the patient's ownphysiological fluids. The injection of this complex reduces, and eveneliminates, a patient's allergic reaction to the specific allergen,while not possessing the side effects accompanying conventional allergytreatments.

Another antibody/antigen complex is taught in EP 426913, published May15, 1991. This application discloses a complex composed of factor VIIIand an antibody termed factor VIII inhibitor. This complex is used totreat hemophiliacs who have become refractory to the injection of factorVIII. These individuals produce a factor VIII inhibitor antibody whichbinds with, and renders factor VIII inactive. Thus factor VIII functionsas the antigen, while the anti-factor VIII functions as the antibody. Itwas demonstrated that the injection of the factor VIII antigen/inhibitorcomplex reduces some patient's refractortness to factor VIII injections.

The antigen/antibody complexes taught in St. Remy et al and the pendingapplication preferably use the antibody from the patient's ownphysiological fluids. This is significant not only because it eliminatesany potential reaction to an antibody prepared from a non-identicaldonor, but more importantly, autologous antibodies are most effectivefor induction of the therapy itself. The presently available methods, astaught by St. Remy et al and the referenced application, of extractingthe antibodies requires painstaking chemical isolation of the antibodyfrom the patient's bodily fluid, such as blood. For example, St. Remy etal discloses isolating the desired antibodies IgG, IgM, IgA, IgE, andIgD by a complex series of steps involving precipitation, dialysis,concentration, chromatography and immunoadsorption.

Thus while pharmaceutical compositions containing antigen/antibodycomplexes may be beneficial in the treatment of certain diseases andconditions, the methods of isolating the desired antibody are complexand time consuming. It is thus desirable to provide a mechanism toreadily prepare pharmaceutical antibody/antigen complexes.

Recent advances in separation technology have presented the opportunityof isolating a specific target population, e.g. cells, proteins, orantibodies without the need of tedious and extensive chemical separationtechniques. For example, various workers have suggested capturingspecific target populations by using filters bearing immunoreactivegroups. Immunoreactive groups are those to which the desired antibodywill selectively bind, typically the antigen. These techniques includefilters prepared from fibers bearing immunoreactive groups, U.S. Pat.No. 3,843,324, issued on Oct. 22, 1974; columns bearing immunoreactivegroup, U.S. Pat. No. 4,252,653, issued on Feb. 24, 1981; and filtercandles bearing immunoreactive groups, U.S. Pat. No. 4,648,974, issuedMar. 10, 1987.

Another recently developed technique for isolating target populations,i.e. antibodies, selected proteins and cells, from a physiological fluidutilizes paramagnetic beads or particles coated with an immunoreactivecompound or agent selective for the desired target population. Examplesof such particles or beads are disclosed in U.S. Pat. Nos. 4,230,685,issued Oct. 28, 1980; 4,554,088, issued Nov. 19, 1985; and 4,628,037,issued Dec. 9, 1986. The use of such particles in the separation oftaught in publications, "Removal of Neuroblastoma Cells From Bone Marrowwith Monoclonal Antibodies Conjugated to Magnetic Microspheres", by J.G. Treleaven, J. Ugelstad, T. Philips, F. M. Gibson, A. Rembaum, G. D.Caines and J. T. Kemshead, The Lancet, Jan. 14, 1984, pages 70-73, and"Immunomagnetic removal of B-lymphoma cells from human bone marrow: aprocedure for clinical use", by G. Kvalheim, O. Sorensen, O. Fodstad, S.Funderud, S. Kiesel, B. Dorken, K. Nustad, E. Jakobsen, O. Ugelstad andA, Pihl, Bone Marrow Transplantation, (1988), volume 3, pages 31-41.

Other references which disclose devices and methods of isolatingspecific target populations are U.S. Pat. Nos. 3,970,518, and 4,018,886both issued to Giaever on Jul. 20, 1976 and Apr. 19, 1977, respectively,4,219,411, issued to Yen et al on Aug. 26, 1980 and 4,710,472, issued toSaur et al on Dec. 1, 1987. Both Yen et al and Saur et al discloseintricate devices for mixing a physiological fluid with Immunoreactiveparamagnetic particles. These immunoreactive paramagnetic particles arecoated with an agent specific for the target population, that is, willselectively bind to the target population cellular members or chemicalspecies. These paramagnetic particles are subsequently captured by amagnetic field while removing the fluid.

The Giaever patents disclose devices and methods of isolating the selectpopulation of cells from a physiological fluid using the immunoreactiveparamagnetic particles. The immunoreactive paramagnetic particles andthe physiological fluid are mixed in a vessel. Once the immunoreactiveagents, i.e. antibody layer, has bonded with the target population cellsor chemical species, the particles are separated from the fluid byactivating a magnetic coil to capture and immobilize the particles andopening a valve to release the fluid from the vessel. The immunoreactiveparamagnetic particles are then transferred to another vessel containinga cleaving agent. This cleaving agent promotes the release of theselected target population from the coated paramagnetic particles.

The above discussed procedures and apparatus allow for the separation ofa target population, whether cells, proteins or antibodies, from aphysiological fluid. The procedures for which these methods are used aretypically those in which it is desired to remove this target populationfrom the physiological fluid prior to returning the fluid to thepatient. For example, the above procedure is useful for removinginfected or tumor cells from a specific tissue, e.g. bone marrow. Themajor drawback to the described procedures and apparatus is thecomplexity of the procedure or apparatus used, or the inability ofthoroughly separating the target population from the remainder of thephysiological fluid. That is, the disclosed procedures and apparatuswere not as concerned with the complete removal of most of thephysiological fluid since the primary focus is the removal of the targetpopulation. Thus there remains the risk of some contamination of thetarget population with extraneous cells or compounds from the fluid.

Further, such procedures and apparatus have the drawback of requiringmultiple containers for performing the isolation, as described inGiaever et al, or require specific knowledge of procedures to insureprevention of contamination of the final product. It thus remainsdesirable to provide for a method and apparatus which would simplify thehandling and isolation of a target population, and specifically anantibody, for the purpose of easily, and safely preparing apharmaceutical composition.

SUMMARY OF THE INVENTION

The present invention overcomes the above described disadvantages byproviding a method, and useful apparatus for the preparation ofpharmaceutical compositions comprising a complex of an antigen componentand the corresponding antibody component in a pharmacologicallyacceptable carrier.

The process of the invention is carried out by isolating the selectedantigen component/antibody component complex in a pharmaceuticallyacceptable carrier in a chamber.

Generally the process of the invention involves preparing apharmaceutical composition of a specific target antibody componentpopulation in a pharmaceutically acceptable carrier from a physiologicalfluid in a chamber having at least one inlet and outlet comprising:

bringing the physiological fluid into contact with a solid phase supportbearing an antigen component selective for the target antibody componentpopulation;

removing the physiological fluid from the chamber;

washing the chamber with an acceptable wash fluid;

draining the washing fluid from the chamber;

supplying a pharmaceutically acceptable eluting solution to the chamber;and

removing the eluting solution from the chamber.

Additionally, the invention relates to a disposable, aseptic single usefluid processing pathway that can be used to obtain a predeterminedamount of an antigen-specific antibody from a patient's physiologicalfluid to treat allergy or autoimmune disease by immunization withautologous or homologous antigen:antibody immune complexes.

Additionally, this invention relates to a method to obtain apredetermined amount of allergen-specific antibody from physiologicalfluid to conduct a course of immunotherapy treatment for an allergicreaction to that specific allergen comprising:

adding a sufficient amount of a physiological fluid and a sufficientnumber of allergen coated particles to bind a predetermined amount ofallergen-specific antibody to an aseptic separation chamber;

incubating said fluid and the allergen coated particles for a sufficientperiod of time to bind the allergen-specific antibody to the allergencoated particles in the chamber;

means to separate the allergen-specific antibody bound to the allergencoated particles from unbound fluid;

means to separate said allergen-specific antibody bound to said allergencoated particles from unbound fluid;

removing the unbound fluid from the chamber;

washing the allergen coated particles to remove nonspecifically adsorbedmaterials in the chamber;

adding an amount of a physiologically acceptable elution solution toelute the allergen-specific antibody from the allergen coated particles,but less than an amount of the elution solution to require concentrationof the recovered allergen-specific antibody;

incubating the allergen-specific antibody bound to the allergen coatedparticles with the elution solution for a sufficient period of time toelute the allergen-specific antibody from the allergen coated particle;

means to separate the allergen-specific antibody from the allergencoated particles in the chamber, to obtain a predetermined amount ofallergen-specific antibody;

adding an amount of a physiologically acceptable neutralizing buffer tothe allergen-specific antibody to obtain a solution having aphysiologically acceptable pH, but less than an amount of theneutralizing solution to require concentration of the allergen-specificantibody,

In accordance with a preferred embodiment of the invention the solidphase support are paramagnetic particles bearing the antigencomponent'selective for the antibody component target population.Specifically, the method of the invention involves preparing apharmaceutical composition by isolating a target antibody componentpopulation from a physiological fluid in a pharmaceutically acceptablecarrier, which isolation is performed in a chamber which is at leastpartially magnetically permeable and includes an inlet and outletcomprising:

mixing the physiological fluid and immunoreactive paramagnetic particlesbearing an antigen component selective for the target antibody componentpopulation;

capturing the immunoreactive paramagnetic particles in magnetic field tohold the paramagnetic particles in the chamber;

draining the fluid from the chamber;

adding a washing fluid to the chamber;

removing the washing fluid from the chamber;

supplying a pharmaceutically acceptable eluting solution to the chamber;and

removing the eluting solution from the chamber.

The invention is also directed to a device for selectively isolating atarget antibody component population from a physiological fluid inaccordance with the described method. This device includes a housingdefining a chamber, which includes at least one inlet and outlet forgaining entrance to the chamber. The housing is preferably formed atleast partially from a magnetically permeable material. The devicefurther includes a platform against which the housing is rested andreleaseably retained. This platform may be positioned in either asubstantially horizontal or vertical position. The platform may alsoinclude one or more magnets located adjacent to housing when positionedon the platform. In accordance with a preferred embodiment, the platformis secured to a support which allows the platform, and thus the housingto be rotated between a substantially vertical to substantiallyhorizontal position.

Additionally, the invention is also directed to a kit comprisingreagents and a disposable aseptic separation device to recover elutedantibody, said kit being used to form autologous or homologousantigen:antibody immune complexes to treat allergy or autoimmunedisease.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be better understood and the advantages willbecome apparent to those skilled in the art by reference to theaccompanying drawings, wherein like reference numerals refer to likeelements in the several figures, and wherein:

FIG. 1 is a prospective illustration of a container assembly inaccordance with an embodiment of the invention suitable for performingthe method of the invention;

FIG. 2A is a front view of a support assembly in accordance with anembodiment of the invention; and

FIG. 2B is a top view of FIG. 2A.

FIG. 3 is a schematic diagram of the present invention in the kitformat, to form antigen allergen-antibody immune complexes to treatallergy or autoimmune disease,

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to a method and useful apparatus, forpreparing a pharmaceutical composition directly from a physiologicalfluid, e.g. blood or plasma. The pharmaceutical compositions prepared inaccordance with the method and apparatus of the invention are thosecompositions useful for treating ailments caused by the reaction of thebody to a specific antigen component.

For the purpose of this invention "antigen component" shall be definedgenerally as any compound or molecule having a chemical site which isrecognized and bound to by an antibody component (as herein defined).Examples of antigen components are those compounds or molecules whichreact with immunoglobulin molecules. Antigen component shall includeantigens in general, and also any other naturally occurring or foreigncompound which invokes an immunological response from the body. Examplesof specific antigen components in accordance with the invention areproteins, carbohydrates, hormones, allergens, and factor VIII, when theadministration of factor VIII to hemophiliacs causes a refractoryresponse.

For the purpose of this invention "antibody component" shall begenerally defined as any compound or molecule which reacts with arecognized chemical site of an antigen component, and in particular animmunoglobulin which the body produces in response to an antigencomponent. Examples of antibody components include those immunoglobulinsproduced in response to viruses, allergens and factor VIII inhibitor,antibiotics, chemicals, or any substance where this response occurs. Inthis regard, antibody component shall include those immunoglobulinsproduced by the body in response to compounds.

Generally, the method of the invention involves selectively isolating adesired target antibody component population from a patient'sphysiological fluid using a solid phase support to which selectiveantigen components have been affixed. This solid phase support willtypically be particles or beads prepared with a coating or layer of anantigen component selective for the desired antibody component which maybe admixed with the physiological fluid. Although, it may be possible touse the chamber walls as the solid phase support. It is generallydesireable to immobilize as much antigen on the particle or bead aspossible to facilitate the capture of the greatest amount of antibody.The antibody components bind to the antigen component forming an antigencomponent/antibody component complex. The fluid and beads are thenseparated, with any unbound materials, e.g. cells, proteins or othercompounds washed away from the particles. The beads can be separated bymagnetic separation if they are paramagnetic or by filtration if theyare not magnetic. The particles are then contacted with an elutionsolution which either causes the disassociation of the antibodycomponent from the antigen component, or cleaves the antigencomponent/antibody component complex from the particles or beads. Thiselution solution is collected and used to prepare the pharmaceuticalcomposition.

Preferred particles are immunoreactive paramagnetic particles or beads.The types of paramagnetic particles useful for the practice of theinvention are typically polymeric spherical particles having diametersfrom about 3 to about 8 microns. These particles are formed with smallamounts of magnetite, which causes the particles to be captured by amagnetic field. However, these types of particles do not retain anymagnetism after removing the field. Generally paramagnetic particles arethose having a magnetic susceptibility per unit volume from about 1×10⁻³to about 1×10⁻² cgs units.

The paramagnetic particles useful for practicing the invention shouldprovide for an adequate antigen component binding surface capacity,while possessing a low non-specific binding characteristic. That is, theparticles should be prepared from a material which ensures an adequatecapacity for retaining antigen component, while also being a materialwhich possesses limited reactivity with compounds or materials otherthan the desired antigen component. Examples of suitable paramagneticparticles are disclosed in many of the above referenced patents, with apreferred paramagnetic particle being that sold under the identificationnumber M450 by the Dynal Company of Great Neck, N.Y. Additionally,paramagnetic Sepharose® (Pharmacia Inc.) beads may be employed.

The precise type of paramagnetic particle used in the practice of theinvention is not critical and will not be described any further herein,so long as the paramagnetic particles possess a desired capacity for thespecific antigen component and are characterized by having a low bindingcharacteristic for compounds, cells or other materials other than thedesired antigen component.

As stated, the particles are prepared with an antigen component coatingselective for the desired antibody component. The resulting particlesare generally referred to herein as immunoreactive paramagneticparticles. The antigen component coating includes that antigen componentto which the desired antibody component normally binds to form acomplex. Examples of antigen components useful for the practice of theinvention include those compounds and substances generally known asallergens, i.e. compounds which produce an allergic response. Anotherexample of a useful antigen component is factor VIII. As described in EP426913, published May 15, 1991, mentioned above, factor VIII functionsas the antigen component for factor VIII inhibitor, which is an antibodycomponent. For a detailed description of the treatment of allergies andfactor VIII refractortness see St. Remy et al and the stated co-pendingapplication, both of which are generally incorporated herein byreference.

In accordance with the method of the invention, the selective isolationof the desired antibody component is performed by bringing aphysiological fluid containing the specific antibody component intocontact with the immunoreactive particles, and preferably withimmunoreactive paramagnetic particles.

The physiological fluid may be obtained directly from the patient orfrom multiple donors. Specifically, the physiological fluid may beblood, plasma, lymphatic fluid, cerebrospinal fluid, or derivativesthereof. The physiological fluid should include a sufficient quantity ofthe desired antibody component. For example, physiological fluid takenfor the purposes of providing a composition to treat allergic responsesshould be taken from patients or donors that have been sensitized tothat specific allergen either as a result of natural exposure or activeimmunization. Similarly, hemophiliacs who suffer from the clinicalconsequences associated with the production of factor VIII inhibitorwill normally have a measurable level of such inhibitor present in theirblood.

The physiological fluid may have been previously subjected to atechnique used to grossly separate various constituents, for examplecentrifugation techniques used to separate larger blood cells fromlighter plasma and proteins, including the desired antibody component.This gross separation may enhance the method of the invention.

In accordance with the preferred embodiment using immunoreactiveparamagnetic particles, these particles are admixed with thephysiological fluid, typically in a container. When the container isemployed as part of a single use kit to treat allergy or autoimmunedisease by immunization with autologous antigen:antibody immunecomplexes, the volume of the container may range from about 0.25 mL to500 mL, but preferably 60 mL of fluid. If homologous antibodies are usedmore than 500 mL of physiological fluid may be required. "Homologous"means derived from the same species e.g. Gamma Guard® (Baxter HealthcareCorporation, Hyland Division). The container is one which is at leastpartially magnetically permeable, i.e. at least a portion of thecontainer is non-metallic. The container is then positioned within amagnetic field to capture and draw the immunoreactive paramagneticparticles towards a side of container. The use of multiple magnetspositioned around the container to generate a strong field may result inthe immunoreactive paramagnetic particles being drawn to more than onecontainer side.

With the container maintained in the magnetic field, the physiologicalfluid is expelled. Typically, this is performed by opening a clamp orvalve which releases the fluid through a line leading out from thecontainer. A pump may be used to draw the fluid out of the container.The container may be positioned to place the outlet line in anorientation to allow for gravity flow of the fluid out of the container.

After the physiological fluid is expelled, an appropriate washingsolution is added to the container to wash away any compounds which havenon-specifically bound or adhered to the container walls or theparamagnetic particles. This typically requires adding saline to thecontainer. The magnetic field is removed and the particles and salineadmixed to ensure proper washing of the particle surfaces.

Subsequently, the container is again placed within the magnetic field todraw the paramagnetic particles against one or more sides, and thesaline, or other suitable washing solution is expelled in a mannersimilar to that used to expel the physiological fluid. This washing stepmay be repeated as necessary. Thereafter, an eluting solution is addedto the container. The eluting solution may either be washed over theparticles maintained within the magnetic field, or the magnetic fieldmay be removed and the particles suspended in the elution solution.

Eluting solutions suitable for the practice of the invention depend uponwhether the antigen component/antibody component complex is to bedisassociated or cleaved entirely from the surface of the particles. Inthis regard the specific eluting solution used is dependent upon thebonding between the antigen component and antibody component or thechemical bond holding the antigen component to the particle surface. Itshould be noted that the eluting solution should not be of the typewhich would attack or damage the target antibody component.

Examples of eluting solutions useful for causing the disassociation ofthe antigen component/antibody component complex, that is the release ofthe antibody component from the antigen component, are disclosed in U.S.Pat. No. 4,740,371, issued to St. Remy et al and in U.S. Pat. No.4,431,560, issued to Lake et al on Feb. 14, 1984, both of whichdisclosures pertaining to elution solutions are incorporated herein byreference. Specifically, elution solutions are disclosed in the Lake etal patent at column 2, line 18 through column 3, line 4. Other usefuleluting solutions include acetic acid, citric acid or glycine. When itis desirable to cleave the antigen component/antibody component complexfrom the particle surface the covalent bond between the antigencomponent and the paramagnetic particles is broken by the use of asuitable technique. For example, the antigen component may be covalentlybonded to the paramagnetic particles via tit sulfide bridges which arereadily cleaved with a reducing agent.

The eluting solution should also be of the type which may be injectedinto a patient, that is a pharmaceutically acceptable solute, or of thetype which is readily altered for injection into the patient. Forexample, when glycine is used, phosphate buffer may added in anappropriate amount to effect neutralization.

The precise type of eluting solution is not critical to the invention solong as it possesses the desired characteristics. Further, the volume ofelution solution used should be substantially equivalent to the finalvolume desired for the pharmaceutical composition. In this regard, itwould be desirable to neutralize the elution solution with a powder orequivalent material, e.g. buffer salts to minimize any increase involume, otherwise the volume of the neutralizing agent must be takeninto account when deciding upon the desired volume amount for thepharmaceutical composition. The volume of eluting solution will besignificantly less than the amount of physiological and washing fluidsused, typically on the order of 10 to 50 fold less, however, the volumecould be smaller.

The incubation time of the eluting solution is from between 5 minutes to24 hours to elute the antibody or antibody-antigen complex.

In the context of a single use kit to treat allergy or autoimmunedisease, the volume of solutions added to the separation chamber orcontainer is important. In particular, one of the features of thisinvention that allows the use of a kit is the ability to recover thedesired antibody in a small volume, thus, eliminating the need toprocess the antibody further by way of concentration, dialysis, etc. Ina full size device, elution volumes range from 0.1 to 5 mL, with thepreferred range of 0.1 to 1.0 mL. The largest factor involved inminimizing the volume of elution buffer used is that if one uses toosmall an elution volume, a significant portion of the total elutionbuffer remains in the beads and thus a significant portion of the elutedantibody becomes entrapped in the beads and cannot be recovered. Withthis constraint in mind, another factor having an impact on the neededvolume of elution buffer is the number of antigen or allergen beads usedto bind the antibody. Another factor to be considered is that whenemploying too small an elution volume the yield of recovered antibodywill suffer somewhat. The loss of yield must be balanced with theminimum quantity requirements for the recovered antibody. The optimalrecovery of eluted anti-factor VIII antibody occurs while using 1 to 2mL of elution buffer with 1-2×10⁹ paramagnetic polystyrene (Dynal) beadsin the 50 mL to 60 mL chamber.

The elution solution can be neutralized with a pharmaceuticallyacceptable neutralization solution such as phosphate or imidazolebuffers. Similarly, as with the elution solutions, small volumes ofneutralizing solution are required to facilitate packaging of thisprocess as a kit. In particular, a small volume of a concentratedneutralization buffer can be employed so that neutralization does notresult in a significant dilution of the eluted antibodies.

In the present invention, we have been able to accomplish neutralizationof 1-2 mL of the 0.2 M glycine buffer, pH 2.5 with as little as 20-50microliters of a concentrated neutralization buffer. Neutralizingbuffers such as Tris, glycyl-glycine or borate may also be used.Additionally, it may be possible to choose a suitable neutralizationbuffer such as glycine or pyrophosphoric acid with a higher pKa whichcould allow the use of an even smaller volume of neturalization buffer.Additionally, a dry lyophilized neutralization buffer may be employed.

Critical elements that will dictate a suitable neutralization bufferwill include such considerations as the "buffering capacity" of thebuffer, the safety and toxicology characteristics of buffer, thesolubility of the buffer salts, and the potential leaching of componentsof the contemplated glass vials in which the buffer will be stored inthe kit form.

Typically, the pharmaceutical compositions prepared in accordance withthe invention will require at least a minimum amount of the antigencomponent/antibody component complex in order to achieve the desiredclinical result. This is independently established for treating eachparticular disease or syndrome. After the minimum requirement for thecomplex is established, the procedure can be standardized to achieve thedesired concentration of the antigen component/antibody componentcomplex in the pharmaceutical acceptable eluting solution. The method isparticularly adaptable to allow individual practitioners to obtain andprepare the desired pharmaceutical composition with minimal effort andtime from a patient's own physiological fluid. This thus allows for animmediate application of the desired pharmaceutical composition to thepatient.

The first step in developing a procedure for a specific clinical needinvolves determining the minimum amount of the desired antigencomponent/antibody component complex. Once this is known, the necessaryamount of antibody components is selected to be greater than or equal tothe molar amount of the minimum amount of the antigen componentpopulation necessary to achieve the desired amount of the complex.Preferably the antibody component is used in a molar excess sinceadditional antibody components will not interfere with the functioningof the composition, nor present harm to the patient, but will ensureadequate coverage of the antigen component.

It should be noted that under certain circumstances it may be desirableto provide for a molar excess of the antigen component rather than ofthe antibody component. For this purpose the following discussedprocedures are followed in a similar manner except for the fact ofallocating a molar excess of the antigen component.

The collection of the desired quantity of the antibody component isdependent upon the concentration of antibody components present in theparticular physiological fluid being used, and the number ofimmunoreactive paramagnetic particles used in the practice of theinvention. That is, once the minimum concentration of the antigencomponent/antibody component complex for therapeutic results isestablished, that volume of immunoreactive paramagnetic particles whichpossesses an adequate capacity for the binding of a sufficient quantityof antigen component to extract either a desired quantity or excess ofthe antibody component from a given quantity of the physiological fluidis used in the described method.

Two specific methodologies have been developed using the method of theinvention for preparing specific antigen component/antibody componentcomplexes in the treatment of allergic reactions and the treatment offactor VIII inhibitor. The first general methodology is disclosed in theincorporated herein patent issued to St. Remy et al on Apr. 26, 1988,U.S. Pat. No. 4,470,371. This patent teaches a composition useful forthe treatment of allergic reactions. Basically, the disclosedcomposition is a complex of the specific antigen or allergen causing theallergic reaction, and the antibody specific for that allergen. In thisregard all teachings and discussions concerning the administration ofthe allergen/antibody complex for the purpose of treating the allergicreaction are incorporated herein by reference, and specifically theteachings in column 3-6.

The major distinction between what is taught in St. Remy et al and thepresent invention concerns the method of isolating the antibody. This isspecifically discussed at columns 7 and 8 under the heading 2, AntibodyPurification. The purification procedure taught by St. Remy et alinvolves an intricate and time consuming purification method usingconcentration on and extraction techniques. The method of the inventionallows for the purification of the antibody components in a simpler moreconcise manner with the use of the immunoreactive paramagneticparticles.

The concentration of the allergen/antibody complex necessary forachieving the desired therapeutic results discussed in St. Remy et al isspecifically taught by St. Remy et al, and particularly at column 5,line 13, under the heading "2. Formation of the composition of theinvention", and at Column 6, line 12, under the heading "Administrationof the compositions", which is also incorporated herein by reference. Asuitable quantity of the immunoreactive paramagnetic particles should beselected to provide for the desired quantity of antibody for complexingwith allergen. In contrast to the above described method, apredetermined amount of antigen or allergen-specific antibody, for usein a kit, is an amount required for a course of treatment that does notrequire concentration or dialysis.

A second methodology is disclosed in the EP 426913, published May 15,1991. This application teaches a composition useful for the treatment ofthe adverse reactions caused in some patients by factor VIII inhibitor.As discussed, some hemophiliacs develop an allergic type reaction to theadministration of factor VIII. This reaction is caused by the presenceof excessive quantities of factor VIII inhibitor. The methodology taughtin this patent application involves the administration of a complex ofthe factor VIII inhibitor and factor VIII. In this regard all teachingsand discussions concerning the administration of the factor VIII/factorVIII inhibitor complex for the purpose of treating the describedcondition are also incorporated herein by reference.

The above described method may be performed with any suitable device,e.g. the device disclosed in the EP 426913, published May 15, 1991.Further, when using immunoreactive particles which are non-paramagnetic,the device may include a filter for trapping the particles as thevarious fluids are being removed.

A preferred apparatus for performing the described method will now bediscussed with reference to the several figures. The apparatus of theinvention includes two parts, container assembly seen in FIG. 1 at 10,and support assembly seen in FIG. 2A and 2B at 12.

Container assembly 10 generally includes a separation chamber 14 havingmultiple inlets and outlets. This separation chamber 14 should be sealed or in some manner closed off to external contamination. Containerassembly 10 is designed to allow a user to perform the above describedprocess entirely within the separation chamber 14. The separationchamber 14 is a generally elongated cylindrical body formed with twoopposing ends 11 and 13. End 11 is provided with the various inlets,while end 13 includes at least one outlet and is formed to taper to thisoutlet, as seen generally as tapered portion 15.

To facilitate the introduction of the various fluids and immunoreactiveparamagnetic particles, the separation chamber 14 is provided with fluidtubes 16 and 18. These fluid tubes 16 and 18 are used to introduce thephysiological fluid into the separation chamber 14, via tube 16, and thesaline or similar washing fluid, via tube 18.

The fluid tubes 16 and 18 extend out from end 11, and are eitherintegrally formed with the separation chamber 14, or are mounted theretoin such a way to provide for a suitable seal. Clamps 20 and 22 arefitted about the fluid tubes 16 and 18, respectively. These clamps 20and 22 are used to seal the respective fluid tubes 16 and 18 after theintroduction of the specific fluid to prevent contamination. Typically,such clamps 20 and 22 may be Roberts, slide or roller clamps.

End 11 of container assembly 10 also includes a vent 7 and septum 9.Septum 9 is used to introduce the immunoreactive paramagnetic particles,not shown, and the elution fluid into the separation chamber 14. Thevent 7 and septum 9 are formed to limit contamination within theseparation chamber 14. In this regard, vent 7 and septum 9 may beone-way valves. Vent 7 may also be an opening covered by filter materialhaving a porosity designed to allow for air flow but to preventcontamination of the separation chamber 14.

Separation chamber 14 includes a single outlet extending out from end13. This outlet is seen as tubing 24. Tubing 24 connects to the mosttapered end of tapered portion 15, and is mounted thereto and integrallyformed with end 13. The combination of the tapered portion 15 and tubing24 defines a funnel like structure to direct fluid out of the separationchamber 14. The tapering of the outlet in this manner minimizes thepotential that fluid will remain within the separation chamber 14. Thisfluid tubing 24 is dimensioned to control the rate of flow out of theseparation chamber 14. Typically, fluid tubing 24 will have an internaldiameter of from about twenty thousandths of an inch to about sixtythousandths of an inch.

As seen in FIG. 1, tubing 24 is formed with a Y-section, seen generallyat 26. This Y-section 26 includes two arms 28 and 30. One arm 28 will beused to expel waste fluid, i.e. the physiologic fluid and any washingfluid. The other arm 30 will be used to direct the resulting compositionof the antibody component, or antigen component/antibody componentcomplex in the eluting solution into an appropriate container, notshown.

In order to facilitate the directing of the fluid from the separationchamber 14 through the appropriate arms 28 and 30 of the tubing 24 atwo-way valve 33 is positioned at the Y-intersection. This two-way valve33 is formed with two separate flow pathways, 29 and 31, through whichfluid is selectively directed to either of two arms 28 and 30. Two-wayvalve 33 is thus operated to direct the flow only through one of thesearms, while limiting the possibility of the physiological or washingfluid entering the other such arm.

In place of the two-way valve 33, clamps, not shown, can be positionedat the top of each of the two arms 28 and 30 to prevent unwanted flow.The opening of either of these clamps would then allow for flow down theappropriate one of the two arms 28 and 30. It may also be desirable toposition a clamp, seen generally at 39, upstream from the two-way valve33. This allows the tubing 24 to be temporarily sealed off from thetwo-way valve 33 during the mixing of the fluids and immunoreactiveparamagnetic particles in the separation chamber 14. A filter assembly,not shown, may be disposed in the arm 30. This filter would function tolimit the passage of immunoreactive paramagnetic particles or anybacteria present in the physiological fluid. Generally, the filter wouldbe of the type having a pore size of about 0.22 millimicrons.

Assembly 10 (FIG. 1) is typically constructed from a flexible material.As stated above, the container, in which the physiological fluid andimmunoreactive paramagnetic particles are placed, is positioned within amagnetic field drawing the immunoreactive paramagnetic particles to oneor more sides of the separation chamber 14. This may be accomplished byplacing the assembly 10 upon a support containing one or more magnets.In this way the immunoreactive paramagnetic particles would be drawntowards that wall of the separation chamber 14 positioned adjacent themagnets.

Referring to a combination of FIG. 1 and FIGS. 2A and 2B, a preferredembodiment of a support assembly 12 will now be described in detail.Support assembly 12 is formed with a platform 32 upon which thecontainer assembly 10 (FIG. 1) is mounted. This platform 32 includes oneor more magnets 34 and a spring biased clasp 36. The magnets 34 areseated in a depression formed in the platform 32 to provide a flatsurface upon which the container assembly 10 (FIG. 1) rests. Magnets 34are preferably a high grade magnetic material prepared fromneodymium-iron-boron with a surface field strength sufficient to capturea majority of the immunoreactive paramagnetic particles. In this regard,the magnets 34 should have a sufficient field reach to extendsubstantially across the separation chamber 14 (FIG. 1). The number ofmagnets 34 will be dependent upon the size of the container assembly 10(FIG. 1).

In another embodiment the magnets have been recessed into the platform.The platform has been modified to accept the placement of a stainlesssteel "keeper" between the disposable chamber and the magnets. The"keeper" is designed to slide in place using a set of grooves milledinto the platform. The purpose of the keeper is to shield the chamberfrom the magnetic field when the chamber and its contents are beingmixed. When the magnetic capture of the paramagnetic beads is desiredthe "keeper" is removed and the chamber is pulled back into a positionnext to the magnets by the spring biased clasp. The "keeper" can easilybe reinserted between the chamber and the magnets when desired.

This magnetic separator is fitted with a variable speed, geared electricmotor, geared rotation axle and a belt connecting the two gears. Theassembly is designed to accommodate the mixing necessary for sampleprocessing and replaces the previously discussed practice of mixing byplacing the chamber on an independent rotator. With the disposablechamber on the separator, the chamber assembly can be rotatedend-over-end on the axle with the rotation driven by the electric motor.

This magnetic separator is fitted with an axle that contains a springbiased gear which can be disengaged if desired and will allow the freerotation of the platform and chamber assembly independent of the motorand drive gears. The axle can be reengaged when the platform has beenrotated to any desired position. This feature allows the rotation of thechamber and platform from the horizontal to the vertical position andvice versa during the processing steps without the need to turn on themotor. This is particularly helpful when the beads have been captured inthe vertical position after the elution solution has been added and theoperator desires to reposition the chamber so that the elution solutionwhich drains to the bottom of the chamber can be recovered while themagnetic beads remain captured on the side of the chamber.

The recessed platform is fitted with tubing restraints which allow thefree ends of the chamber tubing inlet and outlets to be held duringrotation and mixing of the beads with the sample, wash solution orelution.

The above described apparatus leads to the capability to conduct entireoperation of mixing, washing, and elution without ever needing to removethe separation chamber from the magnetic separator. The previouslydisclosed magnetic separator necessitated the removal of the disposablechamber from the magnetic separator for each mixing step and replacementof the chamber on the separator for each magnetic capture step.

The container assembly 10 (FIG. 1) is positioned on the platform 32(FIG. 2a) to place the separation chamber 14 (FIG. 1) at a locationadjacent to magnets 34 (FIG. 2a). This ensures that the magnetic fieldwill pass through substantially all of the separation chamber 14 (FIG.1).

The container assembly 10 (FIG. 1) is held onto the platform 32 (FIG.2a) by the spring biased clasp 36. Clasp 36 is formed with two arms 38and 40 which can be brought into engagement with and hold the separationchamber 14 (FIG. 1) to the platform 32.

This spring biased clasp 36 is designed to releasably grip the containerassembly 10 (FIG. 1) about the separation chamber 14 (FIG. 1). Thisallows the container assembly 10 (FIG. 1) to be repeatedly mounted toand removed from the support assembly 12. This releasable grip isprovided by mounting the clasp 36 in such a way to platform 32 so as tobe reciprocatively biased in a direction towards platform 32 (FIG. 2a).

For example, spring biased clasp 36 (FIG. 2a) may be mounted to slidealong bolts or studs, not shown, extending out from the platform 32.Springs, also not shown, may be positioned about these studs and securedat opposite ends to the platform 32 and the spring biased clasp 36. Inthis manner the clasp 36 may be pulled outward along the studs but whenreleased retract back to the platform 32.

Platform 32 (FIG. 2a) includes at one end a rest 42. Rest 42 is designedand positioned to engage the tapered portion 15 of end 13 when thecontainer assembly 10 (FIG. 1) is being held onto the platform 32.Further, rest 42 (FIG. 2a) is formed with a cut-away 44 through whichtubing 24 extends. The platform 32 is mounted in the support assembly 12to allow for easy rotation from a vertical to horizontal position.

As illustrated, platform 32 (FIG. 2a) is mounted between two sidesupports 46 and 48 to an axis 50. Axis 50 is defined by two threadedbolts 51 and 53 each of which is secured at one end to the platform 32and at the opposite end to one of the side supports 46 or 48. Theplatform 32 is secured to the individual bolts 51 and 53 to allow forfree rotation. Tension is maintained on the platform 32 by mountingsprings 52 and 54 between the platform 32 and washer/nut combinations 56and 58. These washer/nut combinations 56 and 58 are threadably mountedalong the bolts 51 and 53 to ensure that the respective springs 52 and54 remain under tension. This tension ensures that the platform 32 willremain in any desired position about the axis of rotation.

The apparatus 10 (FIG. 1) is operated by first introducing the specificphysiological fluid into the separation chamber 14 (FIG. 1) through thefluid tube 16. The desired quantity of immunoreactive paramagneticparticles is then added to the fluid through the septum 9. The fluidtubes 16 and 18 and tubing 24 are closed off by the clamps 20 and 22,and valve 33, respectively.

At this point the container assembly 10 (FIG. 1) need not be positionedon the support assembly 12 (FIG. 2a), and in order to perform the mixingstep should not be in position adjacent the magnets 34. The containerassembly 10 (FIG. 1) is agitated to thoroughly mix the fluid andimmunoreactive paramagnetic particles.

The container assembly 10 (FIG. 1) is then placed on the supportassembly 12 (FIG. 2a) with the separation chamber 14 (FIG. 1) placedadjacent to the magnets 34 (FIG. 2a). The magnetic field draws and holdsthe immunoreactive paramagnetic particles to that side of the separationchamber 14 (FIG. 1) closest to the magnets 34 (FIG. 2a). Valve 33 isoperated to release the fluid in the separation chamber 14 (FIG. 1) downthrough the arm 28.

A washing fluid, i.e. saline, is then introduced into the separationchamber 14 (FIG. 1) through line 18 after the valve 33 (FIG. 1) isclosed. Again, the container assembly 10 (FIG. 1) is removed from thesupport assembly 12 (FIG. 2a), or otherwise the magnetic filed isremoved from influencing the immunoreactive paramagnetic particles inthe separation chamber 14 (FIG. 1). After sufficiently mixing thewashing fluid and immunoreactive paramagnetic particles, the containerassembly 10 (FIG. 1) is repositioned on the support assembly 12 (FIG.2a). The magnetic field of the magnets 34 (FIG. 2a) again draws andholds the immunoreactive paramagnetic particles against a wall of theseparation chamber 14 (FIG. 1). The valve 33 (FIG. 1) is opened torelease the washing fluid through arm 28.

During the release of both the physiological and washing fluids thecontainer assembly 10 (FIG. 1) is held in the vertical position to allowfor a gravity flow of the fluid out of the separation chamber 14 (FIG.1). After the separation chamber 14 is emptied of the washing fluid, asmall quantity of elution fluid is introduced into the separationchamber 14 via the septum 9. The precise amount of elution fluid shouldbe equivalent to the desired amount of the final amount pharmaceuticalcomposition sought. Typically from about 0.1 milliters to about 5milliters of the elution fluid will be used.

Since only a small quantity of the elution solution is to be used, thecontainer assembly 10 (FIG. 1) may be oriented to expose the fluid toall of the particles, typically by laying the container in a horizontalposition. Alternatively, the elution solution is added to the chamberand mixed end-over-end. The elution solution and particles may also bemixed together by removing the container assembly 10 (FIG. 1) from thesupport assembly 12 (FIG. 2a), and thereby removing the magnetic field.This allows the elution solution to bathe all of the immunoreactiveparamagnetic particles and thus act upon the antigen component/antibodycomponent complex for the purpose of releasing the antibody component orthe entire complex.

After a sufficient amount of time the container assembly 10 (FIG. 1) isagain placed in the vertical position, by rotating the support assembly12 (FIG. 2a) and the valve 33 (FIG. 1) is opened to release the finalproduct through the arm 30. This product is collected and usuallyneutralized by the addition of a suitable neutralizing agent.

A single use kit for performing the described method will now bediscussed with reference to FIG. 3. FIG. 3 shows a schematic diagram ofthe present invention in the kit format. Step 1 of this method involvesadding a sufficient amount of a patient's physiological fluid and asufficient amount of allergen coated paramagnetic particles to bind apredetermined amount of allergen specific antibody to container assembly10 (FIG. 1), that generally includes chamber 14 (FIG. 1) having multipleinlets and outlets.

In step 2 of the method the physiological fluid and allergen coatedparamagnetic particles are incubated for a sufficient period of time tobind the allergen-specific antibody to the allergen in the separationchamber. In step 2 the allergen-specific antibody bound to the allergencoated paramagnetic particles are separated from unbound fluid. In step4 a wash solution is added to the container assembly through an inletport. The wash solution is a physiological saline solution. In step 5 anamount of physiologically acceptable elution solution is added to elutethe allergen-specific antibody from the allergen coated paramagneticparticles, but less than an amount of the elution solution to requireconcentration of the recovered allergen-specific antibody.

The allergen-specific antibody is separated from the retained allergencoated paramagnetic particles and withdrawn through a filter. The elutedantibody solution is withdrawn using a syringe. The syringe is fittedwith a filter and the eluted antibody solution is injected into a vialof neutralizing solution. In step 7 the allergen-specific antibody isadded to a physiologically acceptable neutralizing solution to obtain asolution having a physiologically acceptable pH, but less than an amountof said neutralizing solution to require concentration on of therecovered allergen-specific antibody. In step 8 the allergen-specificantibody is combined with the patient's allergen and the complex isshipped to the allergist to treat allergy by immunization withautologous antigen antibody immune complexes.

The following examples demonstrate the effectiveness of the describedmethod and apparatus in recovering anti-factor VIII antibody (Inhibitor)from a physiological fluid. The beads used in these examples wereprepared with factor VIII covalently bound to their surface. This firstinvolved covalently binding an epoxy moiety to the surface of each beadby treating uncoated beads, sold under the identification number M450 bythe Dynal Company of Great Neck, N.Y., with epichlorohydrin. These beadswere then treated with 1,6,-hexanediamine to yield beads with primaryamino groups. Factor VIII carbohydrate residues were oxidized withsodium periodate to yield aldehyde groups which were then reacted withthe amine derivatized beads to form reversible Schiff's based betweenthe amino and aldehydes groups. These Schiff's bases were reduced withsodium cyanoborohydride to form permanent covalent bonds between theresidues of the amino and aldehyde groups. These beads were used in thefollowing two experiments to demonstrate the ability of the factorVIII-beads to bind anti-factor VIII antibody (inhibitor) from solution.

In lieu of using plasma for these experiments a comparable physiologicalsolution was prepared which consisted of 50 mg/mL of a commerciallyavailable human serum albumin (HSA) and 10 mg/mL of a commerciallyavailable human IgG, both of which are by the Hyland Division of BaxterHealthcare Corporation of Deerfield, Ill., in phosphate buffered saline,which approximated the total protein content of plasma. Radiolabeledanti-factor VIII was added to the HSA and IgG solution at aconcentration of 1.0 μg/mL for each experiment.

The procedures followed for each experiment were as follows:

1. Add solution containing ¹²⁵ I-labeled factor VIII inhibitor at 1μg/mL to the chamber.

2. Add factor VIII bearing bead suspension to the chamber through theseptum port using a syringe and hypodermic needle. The number of beadsper experiment is listed in Table 1 below.

3. Incubate the beads and plasma with end-over-end mixing for 1 hour.The beads must be mixed at least occasionally or the will settle to thebottom of the chamber.

4. Capture beads after the incubation step by placing the chamber on themagnetic separator, i.e. placing the container assembly on the supportassembly in close proximity to the magnets, in the vertical position.Drain the plasma or blood from the chamber to a waste container.

5. Fill the chamber with approximately 40 mm of normal saline washsolution and resuspend the beads by mixing briefly.

6. Recapture beads and drain the saline wash solution from the chamberto waste.

7. Repeat steps 5 and 6 above.

8. Add 2.0 mL of 0.1 M glycine elution buffer, pH 2.5, to the chamber,resuspend the beads and mix for 10 minutes.

9. Capture the beads by placing the support assembly in the horizontalposition and repositioning the chamber in the separator clamps.Accomplishing the bead capture in the horizontal position prevents thebeads and elution buffer from draining to the bottom of the chamberwhile attempting to place the chamber on the separator in the verticalposition.

10. Rotate the separator and chamber into the vertical position andallow the elution buffer to drain to the bottom of the chamber.

11. Repeat steps 8 through 10.

12. Quantitate the factor VIII inhibitor recovered in the elutionbuffer.

13. Results from the two large scale experiments using factor VIII-beadsfor the removal of anti-factor VIII are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        SUMMARY OF THE LARGE SCALE EXPERIMENTS.sup.1,2                                             Experiment                                                                              Experiment                                                          Number 1  Number 2                                               ______________________________________                                        Number of beads                                                                              (1 × 10.sup.9 Beads)                                                                (2 × 10.sup.9 Beads)                         Factor VIII inhibitor                                                         Total bound    13.25 ug    22.57 ug                                           to beads                                                                      Total recovered                                                                              3.22 ug     11.65 ug                                           % Recovered    24%         52%                                                In 1st         3.02 ug     11.10 ug                                           Elution        (94%)       (95%)                                              In 2nd         0.20 ug      0.55 ug                                           Elution         (6%)        (5%)                                              ______________________________________                                         .sup.1 both experiments were conducted under the following conditions:        45 ml volume processed                                                        50 mg/mL HSA &                                                                10 mg/mL human IgG                                                            1.0 ug/mL mouse .sup.125 Ianti FVIII                                          elution with 2.0 mL of 0.1 M glycine, pH 2.5                                  .sup.2 The recovery concentration of the .sup.125 I antifactor VIII, that     is the factor VIII inhibitor during each step of the experiment, that is      the .sup.125 I factor VIII inhibitor recovery on the beads as well as at      each elution step was quantified by using a gamma counter to count the        gamma activity at each level of recovery, which was divided by the total      gamma activity of the .sup.125 I factor VIII inhibitor in the initial         amount used in each experiment.                                          

The large scale experiments using factor VIII-Deeds to remove ¹²⁵I-labeled Hyland anti-factor VIII, that is factor VIII inhibitor, fromsolution demonstrated the capability of the system and apparatus.

Assessment of Antigen Binding Function of Recovered Anti-Factor VIIIAntibodies (Inhibitor)

A series of experiments were conducted to evaluate the antigen bindingfunction of the recovered ¹²⁵ I factor VIII inhibitor. These experimentswere conducted using factor VIII beads and ¹²⁵ I- labeled Hylandmonoclonal anti-factor VIII antibody. Factor VIII beads were incubatedwith 1 μg/mL of the above recovered ¹²⁵ I factor VIII inhibitor in a 50mg/mL human serum albumin and 10 mg/mL human IgG solution for 1 hour.The beads were then washed twice with normal saline and the ¹²⁵ I factorVIII inhibitor was subsequently eluted with 0.1 M glycine, pH 2.5. Theelution solution containing the recovered ¹²⁵ I factor VIII inhibitorwas then neutralized with a suitable buffer and incubated with a secondidentical aliquot of factor VIII bearing beads for 2 hour. The beadswere washed twice with saline and the amount of bound factor VIIIantibodies was determined in a similar manner as described above usingthe gamma activity of the recovered ¹²⁵ I factor VIII inhibitor incomparison to the gamma activity of the initial amount of ¹²⁵ I factorVIII inhibitor.

Utilization of a second aliquot of factor VIII beads as the target forbinding of the recovered factor VIII antibodies made the quantitation ofbound factor VIII antibodies much more reliable than trying to measurethe factor VIII: factor VIII antibodies complexes in solution. Use ofthe target factor VIII beads also makes this a "worst case" measurementsince the kinetics of the factor VIII antibodies binding to a solidphase bead are less favorable binding of factor VIII antibodies tosoluble factor VIII. The results confirm the retention of excellentantigen binding capacity of the recovered antibody. This function isessential to the formation of the factor VIII inhibitor: factor VIIIimmune complexes subsequently used in patient immunization. Binding ofthe anti-factor VIII was also demonstrated to be dependent on the numberof beads used which is consistent with the stated kinetic limitations ofthis model. The data for this experiment is summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                        Summary of the Test Experiments on                                            Rebinding Recovered Factor VIII Inhibitor.sup.12                                       Experiment Number 1                                                                        Experiment Number 2                                     ______________________________________                                        Number of Beads                                                                          (4 × 10.sup.7 Beads)                                                                   ( 8 × 10.sup.7 Beads)                         ANTI-FVIII                                                                    Total % bound to                                                                         24.0           34.8                                                bead aliquot 1                                                                Total % eluted in                                                                        83.2           84.8                                                glycine washes                                                                Total % bound of                                                                         77.0           88.2                                                amount offered to                                                             bead aliquot 2                                                                ______________________________________                                         .sup.1 These experiments were conducted under the following conditions:       2 mL volume processed                                                         50 mg/mL HSA &                                                                10 mg/mL human IgG                                                            1.0 ug/mL mouse 125Ianti FVIII                                                elution with 100 uL of 0.1 M glycine, pH 2.5                                  bead aliquots 1 & 2 for each experiment were of the same size                 .sup.2 Average of triplicate samples                                     

The following examples demonstrate the effectiveness of the describedmethod and apparatus for the recovery of anti-ragweed antibodies fromthe plasma of subjects known to be allergic to ragweed allergenextracts. The beads used in this demonstration were prepared by thefollowing:

1. Uncoated Dynal M-450 beads were purchased from Dynal. Great Neck,N.Y. and modified by the use of epichlorohydrin to have covalentlyattached epoxy residues on their surface.

2. The epoxy derivatized beads were modified by the addition of1,6,-hexanediamine to yield beads with a primary amine group on theirsurface.

3. The amine beads were then mixed with ragweed allergen and1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to facilitate thecovalent coupling of ragweed allergen to the amine groups on the surfaceof the beads,

The procedure is generally the same as previously described, except thatthe ragweed allergen coated beads were substituted for the Factor VIIIbeads and subject plasma was used in the chamber instead of thecomparable physiological solution of 50 mg/mL of human serum albumin and10 mg/mL of human IgG used in the Factor VIII example. Additionally,only a single glycine (0.2M glycine buffer, pH 2.1) elution step wasutilized in this experiment. Two mL of glycine was added.

The elution buffer was neutralized by the addition of solid Tris buffer.

The amount of IgG anti-ragweed antibody present in the neutralizedelution was quantitated by using an enzyme-linked immunosorbent assay(ELISA) for the detection of anti-ragweed IgG. Approximately 0.5 μg ofIgG anti-ragweed was recovered using 2×10⁹ ragweed allergen coated beadsand 40 mL of subject plasma.

The capacity of the recovered anti-ragweed antibodies to form immunecomplexes is also demonstrated by the fact that the antibody is capableof binding to the ragweed allergen coated wells used in the ELISA forthe detection of IgG antibodies. The specificity of the anti-ragweedantibodies can be demonstrated by competitive binding inhibition assayswhich also demonstrate the ability of the recovered anti-ragweedantibody to bind to ragweed allergens.

While the preferred embodiments have been described, variousmodifications and substitutions may be made thereto without departingfrom the scope of the invention. Accordingly, it is to be understoodthat the invention has been described by way of illustration andnot-limitation.

We claim:
 1. A magnetic separator apparatus comprising:a housingdefining a chamber, which includes at least a first inlet and at least afirst outlet for gaining entrance to the chamber; a support platform towhich the housing is releasably mounted, the support platform includingone or more magnets located at a surface of the support platform againstwhich the housing is releasably mounted; a base having at least twospaced apart portions between which the support platform is positioned;one or more arms selectively connected at one location to the supportplatform and at a second location to one of the base portions, with oneof such connections of each of the one or more arms being by means allowing for rotation of the support platform about an axis defined by thearms; and spring biased means associated with the one or more arms tohold the support platform at a location about the axis of rotation. 2.The apparatus of claim 1 wherein the one or more arms are at least asingle shaft connected at one point to the support platform and atanother point to the base, with one of the points of connection beingfreely rotatable about an axis defined by the shaft.
 3. The apparatus ofclaim 1 wherein the base includes two spatially separated beams betweenwhich the support platform is positioned, with the one or more armsbeing first and second shafts connected at one location to the supportplatform and at another location to a respective one of the beams,wherein each of the first and second shafts is mounted at one of thelocations for free rotation about an axis defined by the shaft.
 4. Theapparatus of claim 1 wherein the base includes two spatially separatedbeams between which the support platform is positioned, with the one ormore arms being first and second shafts connected at one location to thesupport platform and at another location to a respective one of thebeams, wherein each of the first and second shafts is mounted at one ofthe locations for free rotation about an axis defined by the shaft andwherein the spring biased means is first and second springs mountedrespectively about the first and second shafts, wherein each of thefirst and second springs remains under tension to bear against thesupport platform.
 5. The apparatus of claim 4 wherein the housing is acylindrical container tapered at one end, with the tapering end in fluidcommunication with a single outlet.
 6. The apparatus of claim 5 whereinthe single outlet includes a two-way valve selectively communicatingwith first and second fluid pathways.
 7. The apparatus of claim 1wherein the support platform includes a spring biased clip whichincludes a portion to bear against and hold the housing to the supportplatform, the clip being resiliently biased in a first direction towardsthe support platform, while being moveable in a second direction awayfrom the support platform to allow placement of the housing between theclip portion and the support platform.
 8. The apparatus of claim 6wherein platform includes a spring biased clip which includes a portionto bear against and hold the housing to the support platform, the clipbeing resiliently biased in a first direction towards the supportplatform, while being moveable in a second direction away from thesupport platform to allow placement of the housing between the clipportion and the support platform.
 9. A magnetic separator comprising:asupport platform to which a housing is releasably mounted, the supportplatform including one or more magnets located at a surface of thesupport platform against which the housing is releasably mounted; a basehaving at least two spaced apart portions between which the supportplatform is positioned; one or more arms selectively connected at onelocation to the support platform and at a second location to one of thebase portions, with one of such connections of each of the one or morearms being by means allowing for rotation of the support platform aboutan axis defined by the arms.
 10. A magnetic separator comprising:asupport platform to which a housing is releasably mounted, the supportplatform including one or more magnets located at a surface of thesupport platform against which the housing is releasably mounted; a basehaving at least two spaced apart portions between which the supportplatform is positioned; one or more arms selectively connected at onelocation to the support platform and at a second location to one of thebase portions, with one of such connections of each of the one or morearms being by means allowing for rotation of the support platform aboutan axis defined by the arms; and spring biased means associated with theone or more arms to hold the support platform at a location about theaxis of rotation.
 11. The separator of claim 10 wherein the one or morearms are at least a single shaft connected at one point to the supportplatform and at another point to the base, with one of the points ofconnection being freely rotatable about an axis defined by the shaft.12. The separator of claim 10 wherein the base includes two spatiallyseparated beams between which the support platform is positioned, withthe one or more arms being first and second shafts connected at onelocation to the support platform and at another location to a respectiveone of the beams, wherein each of the first and second shafts is mountedat one of the locations for free rotation about an axis defined by theshaft.
 13. The separator of claim 12 wherein the spring biased means isfirst and second springs mounted respectively about the first and secondshafts, wherein each of the first and second springs remains undertension to bear against the support platform.
 14. The separator of claim10 wherein the support platform includes a spring biased clip whichincludes a portion to bear against and hold the housing to the supportplatform, the clip being resiliently biased in a first direction towardsthe support platform, while being moveable in a second direction awayfrom the support platform to allow placement of the housing between theclip portion and the support platform.
 15. The separator of claim 13wherein the support platform includes a spring biased clip whichincludes a portion to bear against and hold the housing to the supportplatform, the clip being resiliently biased in a first direction towardsthe support platform, while being moveable in a second direction awayfrom the support platform to allow placement of the housing between theclip portion and the support platform.
 16. A method of preparing apharmaceutical composition of a specific target antibody componentpopulation in a pharmaceutically acceptable carrier from a physiologicalfluid using paramagnetic particles bearing an antigen componentselective for the target antibody component population in combinationwith the magnetic system of claim 1 comprising:supplying a quantity ofthe paramagnetic particles to the housing chamber; introducing thephysiological fluid into the housing chamber; mixing the paramagneticparticles and the physiological fluid; removing the physiological fluidfrom the housing chamber; washing the housing chamber with an acceptablewash fluid; draining the wash fluid from the housing chamber; providingthe housing chamber with a pharmaceutically acceptable eluting solution;mixing the paramagnetic particles and eluting solution; and removing theeluting solution from the housing chamber.
 17. A method of preparing apharmaceutical composition of a specific target antibody componentpopulation in a pharmaceutically acceptable carrier from a physiologicalfluid using paramagnetic particles bearing an antigen componentselective for the target antibody component population in combinationwith the magnetic system of claim 4 comprising:supplying a quantity ofthe paramagnetic particles to the housing chamber; introducing thephysiological fluid into the housing chamber; mixing the paramagneticparticles and the physiological fluid to form an antigen component andantibody component complex; removing the physiological fluid from thehousing chamber; washing the housing chamber with an acceptable washfluid; draining the wash fluid from the housing chamber; providing thehousing chamber with a pharmaceutically acceptable eluting solution;mixing the paramagnetic particles and eluting solution; and removing theeluting solution from the housing chamber.
 18. The method of claim 17further including a step of orientating the housing chamber in asubstantially horizontal orientation prior to the steps of removing thephysiological fluid and removing the eluting solution.
 19. The method ofclaim 17 further including the step of neutralizing the eluting solutionafter removal from the housing chamber.
 20. The method of claim 17wherein the selected eluting solution is of the type to cause saidantigen component and antibody component complex to cleave from saidparamagnetic particles.
 21. The method of claim 17 wherein the selectedeluting solution is of the type to cause said antibody component tocleave from the antigen component bound to the paramagnetic particles.22. The method of claim 17 wherein the first and second steps of mixingare performed by first removing the housing chamber from the supportplatform and second by shaking the housing chamber to admix theparamagnetic particles and the respective solutions, and subsequentlyaffixing the housing chamber to the support platform.
 23. The method ofclaim 17 wherein the first and second steps of mixing are performed byrotating the support platform with the housing chamber supportedthereon.